JP2001506979A - Soluble protein - Google Patents

Abstract

The invention includes deleting codon segments from DNA expressing a native protein (e.g., PrPSc) in order to obtain a shorter, soluble protein which mimics characteristics of an insoluble native (e.g., PrPSc) protein. Soluble proteins of the invention are characterized by: (1) having less amino acids than the full length native protein; (2) having a higher degree of solubility than the native protein; (3) retaining the basic biological characteristics of the native protein such as (a) not being subject to enzymatic digestion and (b) causing disease. Soluble proteins of the invention are obtained by providing a DNA sequence which encodes a native protein and systematically removing codons, making copies of the shortened versions of DNA which are then expressed to provide the shortened proteins. The shortened proteins are then tested for solubility. Soluble proteins are then further tested to confirm that they retain the biological characteristics of the native protein. The soluble form can also be created by adding amino acids, binding a hydrophilic moiety to the native protein or combinations of deleting, adding, and binding hydrophilic moieties to the protein.

Description

DETAILED DESCRIPTION OF THE INVENTION                               Soluble protein                                Field of the invention   The present invention relates generally to the field of molecular genetics, and in particular, to solubilize insoluble proteins. And soluble proteins and derivatives thereof.                                Background of the Invention   Some naturally occurring proteins are insoluble and are associated with certain disease states It is known. This includes beta amyloid protein associated with Alzheimer's disease Lewy or Pick bodies associated with plaques, Parkinson's disease and Pick's disease And associated with Creutzfeldt-Jakob disease (CJD) and other neurological disorders Prion protein (PrP^Sc). The mammalian PrP gene is a soluble, non-disease Disease type PrP^CIs insoluble or diseased PrP^ScExpression of proteins that can be converted to I do. In addition, multiple myeloma, thyroid cancer, and Congo red dye-stained vessels There are also a number of systemic diseases involving protein aggregation, including disorders.   PrP^CFrom PrP^ScUnderstanding the conversion to is that bovine prions are transmitted to humans, The possibility that it caused mutant Creutzfeldt-Jakob disease (vCJD) (G. Chazot et al., Lancet 347, 1181 (1996); R.G. . Will et al., Lancet 347, 921-925 (1996)). PrP^ScEven after cutting the N-terminal of It has previously been shown that the infectivity of pea is not lost (S.B. Prusiner et al., Bioch. emistry 21, 6942-6950 (1982); S.B. Prusiner et al., Cell 38, 127-134 (1984)), Correspondingly, PrP^ScOf the N-terminus of PrP^ScConversion to happened (M. Rogers et al., Proc. Natl. Acad. Sci. USA 90, 3182-3186 (1993)). However Any type of PrP^Sc(Ie, disease-inducible proteins) are also insoluble. This Is insoluble, which makes analysis difficult and binds to this protein. Efforts to produce antibodies have been greatly hindered. In addition, the protein Soluble type (PrP^C) Is (1) PrP^Sc(2) Diseases that are digested by enzymes that do not digest It is known not to provoke.                                Summary of the Invention   Because some proteins associated with disease are insoluble, structural analysis of such proteins should be performed. And making antibodies specific for these proteins . The present invention provides soluble forms of such proteins. This is (1) Systematically remove segments of white matter-encoding DNA or (2) add DNA To express the modified protein, or (3) hydrophilic side chains or other Chemically modifying insoluble proteins, such as by adding amino acids with hydrophilic groups, And so on. The resulting protein is (1) soluble and (2 ) Not digested by enzymes that digest the natural soluble form, and (3) its biological Retains function (eg, retains abnormal properties associated with the disease). these Soluble soluble forms of the proteins of the present invention include modified soluble proteins and insoluble native proteins. It is useful for producing antibodies that selectively bind to white matter. This soluble protein is Effect on white matter, that is, the ability of a drug to eliminate the abnormal properties of a disease-associated protein It can also be used to screen drugs for power.   Natural PrP^ScThe soluble form of a naturally occurring insoluble protein, such as the soluble form of Manufactured by the methods described herein. Of insoluble protein in its native form Although other methods can be used to obtain the soluble form, the present invention has been produced in other ways. And soluble proteins of the type described herein. The general method is First, a protein expressed as an insoluble form or an insoluble protein associated with a disease of the nervous system. Determining the DNA sequence encoding one of the proteins that is later converted to white matter including. Thereafter, a copy of this DNA sequence is made. Individual copy or copy The group is used to remove a portion or portions of the DNA. Of course, this Removal is performed on codon segments, and each other is removed because different codon sequences have been removed. Multiple different DNA sequences are provided. Subsequently, these various shortened sequences were expressed. To obtain a plurality of different proteins. The solubility of the various proteins obtained is tested. So Followed by soluble proteins, usually by enzymes that do not digest insoluble proteins. Test for other activities such as digestibility. This protein is soluble and If it is resistant to bioavailability, further biological activity can be tested. For example, this protein In animals that normally develop disease when inoculated with insoluble native protein Can be used to If the inoculated animal develops this protein, (1) soluble; (2) not subject to enzymatic digestion; (3) disease Ability to trigger And / or the ability to produce antibodies that bind to insoluble native proteins And can be characterized as having biological activity.   It is an object of the present invention to provide a protein which is insoluble in its native form, An object of the present invention is to provide a soluble protein which retains its characteristic characteristics.   A more specific purpose is to use the insoluble natural PrP to induce disease.^ScBiological Retains the characteristics, PrP^ScSoluble PrP that produces antibodies that bind to^Sc(Soluble Prio ).   Another object of the present invention is to systematically remove or add DNA segments to produce A truncated sequence that expresses a soluble form of the disease-causing protein that is insoluble in It is to provide a method for obtaining an extended sequence.   Another object of the present invention is to convert insoluble native proteins into soluble Providing a protein that converts to a form, thereby treating a disease associated with an insoluble protein The purpose is to provide a useful protein.   Another object of the present invention is to use the soluble protein of the present invention to produce natural proteins. By providing an antibody that binds to an insoluble form and a method of producing such an antibody is there.   Another object of the invention is the ability of the protein to bind in vivo to agglutinating forms of the protein. Power, and as a result, delays, reverses, or completely aggregates disease-related proteins. Forming a complex to be stopped, by providing a method for producing a soluble protein is there.   Another purpose is that antibodies produced using the soluble protein of the present invention bind to the surface. An object of the present invention is to provide an analysis device including a support that has been provided.   The advantage of the present invention is that soluble proteins can be easily analyzed for their structure compared to insoluble proteins. This means that antibodies can be easily prepared.   A feature of the present invention is that the soluble protein of the present invention is an important organism of an insoluble natural protein. It retains the biological function.   Another feature is the use of soluble proteins with abnormal properties associated with the disease, If we could screen for a drug that converts that protein to a non-disease structure That is to say.   These and other objects, advantages, and features of the present invention are described herein. Will be apparent to those of ordinary skill in the art.                             BRIEF DESCRIPTION OF THE FIGURES   Figure 1 shows PrP^ScShowing the need for proteins in each region to support the formation of PrP 1 is a map of a region of a secondary structure of a protein expressed by a gene.                         Detailed description of preferred embodiments   Before describing the present soluble proteins and methods for producing the proteins, the present invention is described. Is not limited to a particular protein, method, antibody, or process. However, it is necessary to understand that there is a possibility of modification. Also, the scope of the present invention is , The terms used herein, limited only by the appended claims Is only used to illustrate a particular embodiment and is intended to be limiting. It is necessary to understand that there is no.   As used in this specification and the appended claims, the singular forms “a”, “an” and “a” And "the" indicate that the context does not clearly indicate otherwise. Includes the plural of the subject. Thus, for example, “protein” Mixture and a number of proteins, and "an antibody" Body and mixtures thereof, the "the method" being described herein. One or more methods or steps.   Publications mentioned herein should not have been disclosed prior to the filing date of the present application. Provided only to indicate Any disclosure herein is based on prior art. Concluded that the inventor had no right to advance the date on which this disclosure was made. Should not be.   Unless defined otherwise, all technical and scientific terms used herein are Meanings commonly understood by those skilled in the art to which the invention belongs Have. The practice or testing of the present invention will be similar or equivalent to those described herein. Although any methods and materials can be used, preferred methods and materials are described herein. It is listed. All articles cited in this specification are cited in connection with that article. For the purpose of disclosing and describing certain aspects of the present invention, Incorporated in the specification.                                   Definition   The term "soluble" refers to the property of a compound with respect to its ability to mix in a liquid Especially in water, aqueous salts, mixed solvents or liquids such as surfactant solutions. The ability to form a single mixture, a solution that is not just a suspension. Book The solubility of a protein according to the invention can be defined using various parameters. , Those parameters can vary depending on the particular protein for which solubility is to be measured. one In general, insoluble native proteins associated with disease states include water, saline, and mild Insoluble in various surfactants (ie, those that do not denature proteins) The soluble proteins of the present invention are soluble in one or all of these. further Even if 100,000 g of the soluble protein of the present invention is added to a solution for 1 hour, it remains in the supernatant. You. Protein PrP of the present invention^Sc106 is the protein or native PrP^ScDo not denature Soluble in mild surfactant aqueous solutions. PrP^Sc106 mild surfactant , And centrifuged at 100,000g for 1 hour.^Sc106 remains in the supernatant. Modification In confirming the solubility of the protein, various different surfactants can be used. interface One suitable example of an activator composition is 0.5% sodium deoxycholate; Triton X-100 and 0.5%, 1.0% or 2.0% sarkosyl as required Combinations are included. Protein PrP^Sc106 can be used at any of these three concentrations. It is also soluble in lucosyl. Includes salt in addition to surfactant in solvent It is preferred to maintain the pH in the range of 5 to 9, but more preferably 6 to 8, more preferably And preferably maintained at about 7.5. Examples of salts that can be used include 10 mM Tris-HCl, pH 7.5. 100 mM NaCl (sodium chloride); or 1 mM EDTA. Concentration from 1 mM to 1 Other salts in the range of 00 mM can also be used.   As used herein, the term "insoluble" is used to describe the properties of a compound Is not particularly mixed with the liquid, does not dissolve in the liquid to form a uniform mixed solution, In particular, proteins that cannot be dissolved in water or aqueous salt or surfactant solutions This is for explaining the characteristics. Insoluble natural proteins of the type described herein are , A surfactant whose protein does not denature water, water and salt, water and its protein, Or 100,000 g when in water, salt and detergent that does not denature the protein When given for 1 hour, if the protein is not present in the supernatant, it is called insoluble.   Terms such as "digestible" and "enzymatically digested" are interchangeable herein Used under conditions that enzymes normally expect to cleave proteins. When contacted with two or more fragments, preferably multiple fragments (i.e. (Three or more).   One or more specific enzymes used, the time the enzymes are in contact with the protein, And other conditions such as temperature and pressure will vary depending on the particular modified protein being tested. You. When performing digestion tests on proteins expressed by modifying the PrP gene, Using thease K, at atmospheric pressure, a temperature in the range of more than 0 ° C and less than 100 ° C, more preferably Or about 15 ° C to 45 ° C, and more preferably about 37 ° C for about 10 minutes to 2 hours. Box, more preferably 20 minutes to 1 hour, and even more preferably about 30 minutes. Preferably, Kase K is contacted with the protein. The protein is processed at about 37 ° C for about 30 minutes. When denatured upon contact with thease K, the protein is the natural Pr P^ScDoes not possess the biological properties of the protein. If the protein is not digested, Furthermore, if the protein is tested for its ability to induce disease, it can be classified as a protein of the present invention. May be   The terms "not digestible" and "not enzymatically digested" are used herein to refer to Used interchangeably, the protein is not cleaved when the protein is contacted with the enzyme. Means that In particular, the term refers to the fact that enzymes normally cut proteins. Contact with the protein under unusual conditions, the protein remains Means   Terms such as "PrP protein" and "PrP" are used interchangeably herein, and Infectivity known to cause disease (sponge-like encephalopathy) in animals and animals Particle type PrP^ScInfectious PrP, and under appropriate conditions^ScNon-infectious PrP that is converted to a form^C Means both.   “Prion”, “prion protein” used interchangeably herein And "PrP^ScTerms such as "protein" refer to the infectious PrP of the PrP protein.^ScMeans type, Abbreviation of the words “protein” and “infection” Particles are PrP encoded by the PrP gene^ScIf the molecule is not 100% , Mainly including. Prions are different from bacteria, viruses, and viroids. well-known Prio Infected animals include scrapes, a contagious degenerative disease of the nervous system of sheep and goats. P, plus bovine spongiform encephalopathy (BSE) or mad cow disease, and feline spongiform brain in cats Includes those that cause the disease. Four prion diseases known to affect humans Is (1) Kuru, (2) Creutzfeldt-Jakob disease (CJD), (3) Gerstmann's Have Troisler-Scheinker disease (GSS) and (4) fatal familial insomnia (FFI) . As used herein, prions may be present in any animal used, particularly humans and livestock. All or any of these diseases or any other Including prions in the form.   As used herein, the term "PrP gene" refers to the rice issued October 15, 1996. Genetic material that expresses the protein shown in FIGS. 2-4 of US Pat. No. 5,565,186, "Pathogenic Mutations and Polymorphisms" polymorphisms and variants as listed herein under the subtitle “lymorphisms”). Show a difference. The term "PrP gene" generally refers to any form of prion protein Means any gene of any species that encodes Of the widely known PrP sequence Some are Ga, which are incorporated herein by reference to disclose and describe the sequences. briel et al., Proc. Natl. Acad. Sci. USA 89: 9097-9101 (1992). The PrP gene includes any of the "host" and "test" animals described herein. Can be from any animal, and some or all of the polymorphisms and The term is recognized to include other yet undiscovered PrP genes. The protein expressed from such a gene is PrP^C(Non-disease) or PrP^Sc(disease ) Type.   For "standardized prion preparations", "prion preparations", "preparations", etc. The terms are used interchangeably herein and refer to a gene that is substantially identical for a prion. A mammalian brain tissue containing the substance, for example, exhibiting symptoms of prion disease; (2) truncated endogenous prion protein residue (3) It has a high copy number of a prion protein gene from a genetically different species Or (4) a genus of a genetically different species from the truncated endogenous prion protein gene A hybrid derived from a set of mammalian brain tissues that is a hybrid with an on-protein gene. Prion (PrP^Sc). Standardized prion products Is obtained Mammals as a result of prion inoculation and / or Due to the genetically modified nature of the high copy number of white matter genes, Because of the onset of the disease, it exhibits clinical symptoms of abnormal functioning of the central nervous system.   The term "artificial PrP gene" as used herein refers to "chimeric PrP gene". And, when included in the genome of the host animal (eg, mouse), Prions that only infect other test animals, such as humans, cattle, or sheep Other recombinant genes that render the animal susceptible to infection by Including. In general, artificial genes contain one or more (but not all) of the natural sequences, (Generally less than 40) codons are different codons, preferably genetically different mammals ( Genetically altered by being replaced by the corresponding codon It contains the codon sequence of the selected mammalian PrP gene. Genetically modified mammals are Used to analyze prion samples that traditionally only infect other mammals . Examples of artificial genes encode the sequences shown in FIGS. 2, 3, and 4 of 5,565,186. All mouse codons are human, bovine, or sheep At the same relative position, provided that they are not replaced by different codons Human, bovine, and sheep shown in these figures replacing certain mouse codons Is a gene having one or more different substitution codons selected from the following codons: Man The engineered PrP gene contains not only codons from genetically It is not associated with the type PrP gene, but when inserted into an animal, it is usually a genetically different species. Codons that make the animal susceptible to prion infection that only infects the product And may also include codon sequences.   "Chimeric gene", "Chimeric PrP gene", "Chimeric prion protein gene" Terms such as are used interchangeably herein, where one or more codons are Corresponding codons from genetically different test animals such as cattle, cattle, or sheep Artificially produced, including the codons of a host animal, such as a mouse, replaced by Means a gene. In one particular example, the chimeric gene is a host mammal (eg, U.S.A.) containing the start and stop sequences (N- and C-terminal codons) of the PrP gene, Nucleotides of the corresponding part of the PrP gene of the test mammal of the second species (eg, human) Includes code array. When inserted into the genome of a host mammal, the chimeric gene Kind It is usually susceptible to prion infection, which infects only mammals of the second species. Ki The mel gene MHu2M contains the start and stop sequences of the mouse PrP gene and the expressed The corresponding human, which differs from the mouse PrP gene in that the protein Includes non-terminal sequence regions replaced by sequences.   The term "prion-related genetic material" activates the ability to infect prions. It is intended to include any genetic material conferred on an object. So for this The terms “PrP gene”, those terms are defined herein, “artificial PrP gene” Offspring, `` chimeric PrP gene '' or `` truncated PrP gene '' Includes those modifications that allow it to infect prions. Standardized prion preparation All animals become infected with the same type of prion, and All show substantially the same genetic material for prions, as shown by the symptoms of staining. Produced using animals that carry them.   The terms "host animal" and "host mammal" are naturally occurring within that animal. The genome is genetically and artificially manipulated to contain genetic material that does not Used to indicate an object. For example, in host animals, the PrP gene is deleted, Enabled mice, hamsters, and rats are included. Prion protein in host To produce antibodies. Antibody-producing cells should be stored in a phage library. It is a source of genetic material to make. Other host animals include native (PrP) genes or Or the insertion of an artificial gene or the insertion of a native PrP gene from a test animal of a genetically different species. It is possible to have the gene altered by the insertion.   The terms “test animal” and “test mammal” refer to the PrP gene of the host animal and the test animal. Genetically different from the host animal in that it differs from the PrP gene of the test animal Used to indicate animals. Test animals are generally those that are susceptible to infection. Perform analytical tests to determine if lions are present in a given sample. Any animal you want. For example, test animals include humans, cows, sheep, pigs, and horses. Cats, cats, dogs, or chickens that usually only infect test animals. May be trying to determine if the lion is present in a particular sample There is.   The terms “genetically distinct animal” and “genetically distinct mammal” are A test animal of another species is 17 or more codons, preferably 20 or more codons, and Most preferably, the sequence comprises the native PrP codon sequence of the host animal, differing by 28-40 codons. Used to indicate an object. Therefore, the mouse PrP gene is human, bovine, Or a genetically different species for the sheep PrP gene, but Genetics are not genetically distinct.   Terms such as "cut PrP protein gene" and "disrupted PrP gene" It is used interchangeably in the textbook and the gene is modified to be non-functional (eg, Means the endogenous PrP gene with addition and / or removal of nucleotides. Non-machine Examples of functional PrP genes and methods for producing them are described in Buerer, H. et al. Normal development of mice lacking cell surface PrP protein elacking the neuronal cell-surface PrP protein) ”Nature 356, 577-582 (19 92) and Weisman (International Publication No. 93/10227). Remove genes The methods of exclusion are described in Capecchi, Ce, all of which are incorporated herein by reference. ll 51: 503-512 (1987). Preferably, both alleles of the gene are Destroyed.   Terms such as "hybrid animals" and "transgenic hybrid animals" Is used interchangeably herein to delete a truncated endogenous prion protein gene. The first animal with (1) chimeric or artificial PrP genes, or (2) genetically By crossing with a second animal containing any of the PrP genes from another animal Means the animal obtained. For example, hybrid mice are Mouse with offspring and (1) human PrP gene (possibly present in high copy number) Or (2) by crossing with a mouse containing the chimeric gene. The term hybrid refers to a plant whose progeny usually inherit only genetically another species. Includes two hybrid inbred offspring as long as they are susceptible to Lion infection , Including any progeny of the hybrid. Hybrid animals are inoculated with prions and Cells for producing hybridoma for producing monoclonal antibody of the invention Can be used as a source.   Terms such as "susceptibility to infection" and "susceptibility to prion infection" are used herein. Are used interchangeably and are usually genetically infectious only in test animals of a different species. Transgenic or hybrid that develops disease when inoculated with lion The test animals are shown. This term refers to the human prion without the chimeric PrP gene. Infection, but the presence of the chimeric gene makes it more susceptible to human prion infection. Transgenic mice such as Tg (MHu2M) Used to represent brid animals.   "Antibody" means an immunoglobulin protein capable of binding an antigen. Honcho Antibodies used in the textbook include whole antibodies and epitopes, antigens of interest Or any antibody fragment capable of binding to an antigen fragment (eg, F (ab ')_Two, Fab ', F ab, Fv).   The antibody of the present invention comprises PrP^ScImmunoreactivity with the protein or the soluble protein of the present invention or Are immunospecific, and thus specifically and selectively bind to them. Heaven Shire PrP^ScAnd antibodies that are immunoreactive and immunospecific with the proteins of the invention are preferred. No. PrP^ScAnd antibodies against the soluble proteins of the invention are preferably immunospecific , Ie, does not substantially cross-react with related substances. The term "antibody" Although all types of antibodies (eg, monoclonal) are included, the antibodies are preferably Produced using the phage display method described herein.   A “purified antibody” is a protein that is naturally rich in other proteins, carbohydrates, and lipids. Means the antibody that has been excluded. Such antibodies are native PrP^ScProtein and "Selectively bind" to the soluble protein (or antigenic fragment thereof) of the present invention. That is, it does not substantially recognize or bind to other unrelated antigenic molecules. The present invention The purified antibody is preferably a protein of the present invention and a specific species of PrP^ScBoth protein Immunoreactive and immunospecific, more preferably native human PrP^ScAnd Seki It is immunospecific for a series of soluble proteins of the invention.   An “antigenic fragment” of a PrP protein is a portion of such a protein, which A part capable of binding.   "Specifically binds" refers to a specific polypeptide, ie, PrP^ScProtein High binding strength and / or affinity of the antibody for the quality epitope means. Binding of the antibody to an epitope on this specific polypeptide is preferred. Or associated with all other epitopes, especially the specific polypeptide of interest Doing Identical epitopes that may be present within the molecule or in the same sample. Stronger than antibody binding, for example PrP^CPrP stronger than denatured fragment of^ScJoin to Therefore, by adjusting the binding conditions, the antibody is almost completely PrP^CWith denatured fragments of But not PrP^ScCombine with Antibodies that specifically bind to a polypeptide of interest include Possibility of binding weak but detectable levels to other polypeptides (Eg, 10% or less of the binding to the polypeptide of interest). So Weak binding, such as, or background binding, for example, Specific for the compound or polypeptide of interest, such as by using Antibody binding can be easily distinguished. Generally, natural in situ Type PrP^ScAgainst 10⁷Mol / L or more, preferably 10⁸Binding with binding affinity of mol / L or more The antibody of the present invention^ScIt is said to bind specifically to Generally, the affinity is 1 0⁶Antibodies with a binding affinity of mol / L or less are the usual The method is not useful in that it does not bind to the antigen at detectable levels.   "Detectably labeled antibody" "Detectably labeled anti-PrP" or "Detectable Anti-PrP fragment labeled with an antibody (or binding specificity) Antibody fragment). The detectable label is usually due to a chemical bond. However, when the label is a polypeptide, it can be They may be combined. Methods for producing detectably labeled proteins are well known in the art. Is knowledge. The detectable label can be selected from a variety of labels known in the art. , Usually radioisotopes, fluorophores, paramagnetic labels, enzymes (eg, horseradish peroxy) Or a detectable signal (eg, radioactivity, fluorescence, color) Or other components or components that emit a detectable signal when the label is exposed to a substrate. Is a compound. Various detectable label / substrate pairs (eg, horseradish peroxy Dase / diaminobenzidine, avidin / streptavidin, luciferase / Luciferin), methods for labeling antibodies, and methods for using labeled antibodies are well known in the art. (For example, Harlow and Lane (Antibodies: A Laboratory Manual Body: Laboratory Manual) (1988) Cold Spring Harbor Laboratory Press, Cold Sp ring Harbor, NY).   As used herein, "treatment", "treat" and like terms generally refer to a desired drug. It means obtaining a physical and / or physiological effect. This effect can be caused by disease or May be prophylactic in the sense of completely or partially preventing its symptoms, and / or Or partially or completely cure the disease and / or adverse effects caused by the disease Can be therapeutic in the sense that As used herein, "treatment" refers to a mammal, Especially including any treatment of human disease; and (A) In patients who have a predisposition but have not yet been diagnosed with the disease, Prevention of the onset of the disease; (B) inhibiting the disease, ie, stopping its progression; or (C) Alleviating the disease, ie, inducing regression of the disease. The present invention is aimed at treating patients with infectious prions, especially bovine spongy Encephalopathy; Creutzfeldt-Jakob disease; Fatal familial insomnia or Gerstmann's disease PrP leading to central nervous system diseases such as Troisler-Scheinker disease^ScInfected chicks The goal is to treat the disease.   Abbreviations used herein include the following: CNS is the central nervous system; BSE is bovine spongiform encephalopathy; CJD for Creutzfeldt-Jakob disease; FFI is fatal familial insomnia; GSS for Gerstmann-Straussler-Scheinker disease; Hu is a human; HuPrP is a human prion protein; Mo is a mouse; MoPrP is a mouse prion protein; SHa is a Syrian hamster; SHaPrP is Syrian hamster prion protein; Tg is transgenic; Tg (SHaPrP) is a transgenic mouse containing the Syrian hamster PrP gene ; Tg (HuPrP) is a transgenic mouse containing the complete human PrP gene; Tg (ShePrP) is a transgenic mouse containing the complete sheep PrP gene; Tg (BovPrP) is a transgenic mouse containing the complete bovine PrP gene; PrP^ScIs the scrapie isoform of the prion protein; PrP^Sc106 is soluble in mild detergents and consists of residues 89-140 and 177-230 Scrapie isoform of prion protein; PrP^CIs a common normal prion protein isoform contained in cells; MoPrP^ScIs the scrapie isoform of the mouse prion protein; MHu2M differs from mouse PrP in nine codons by the corresponding human sequence in the mouse A mouse / human chimeric PrP gene in which one region of the PrP gene has been substituted; Tg (MHu2M) mouse is a transgenic mouse of the present invention containing the chimeric gene MHu2M . MHu2MPrP^ScIs the scrapie isoform of the human / mouse chimeric PrP gene; PrP^CJDIs the CJD isoform of the PrP gene; Prnp^0/0Are alleles of both endogenous prion protein genes, such as the MoPrP gene Deleting children; Tg (SHaPrP^{+ / 0}) 81 / Prnp^0/0Is a specific transgenic mouse expressing SHaPrP Line (81), + / 0 represents heterozygosity; Tg (HuPrP) / Prnp^0/0Is a mouse with the human prion protein gene (HuPrP) and endogenous Obtained by crossing with mice in which both alleles of the prion protein gene have been disrupted Hybrid mice obtained; Tg (MHu2M) / Prnp^0/0Is a mouse with chimeric prion protein gene (MHu2M) By crossing with mice in which both alleles of the sex prion protein gene have been disrupted The resulting hybrid mouse. FVB is a standard inbred mouse that is often used for the production of transgenic mice. Is. This is because the eggs of FVB mice are relatively large, and microinjection of exogenous DNA This is because the action is relatively tolerable.                                   Overview   Retains disease-related anomalous properties of native protein but becomes soluble upon modification A modified protein is provided. Modifications include addition or substitution of amino acids, All or none of the amino acid deletion or the binding of another molecule to the native protein This can be included. One preferred embodiment of the present invention relates to insoluble native proteins and Shorter versions with similar characteristics, i.e., retain the abnormal properties associated with the disease To obtain a soluble protein, a codon segment is extracted from DNA expressing the native protein. It is an amino acid sequence shorter than the native sequence, obtained by deletion. Book Features of the soluble proteins of the invention include: (1) including structural modifications of the full-length native protein; (2) More soluble than native protein; (3) (a) not enzymatically digested, and (b) disease Retains the basic abnormal biological characteristics of the native protein, such as That is.   The soluble protein of the present invention provides a DNA sequence encoding a native protein, Obtained by specifically removing codons. Make a copy of the shortened DNA, To provide a truncated protein. Thereafter, the solubility of the shortened protein is tested. Then, test the soluble protein to retain the biological properties of the native protein Make sure that   Similarly, other modified soluble proteins of the invention can be prepared and tested. . For example, a specific codon may be replaced with a polar amino acid having hydrophilic properties (eg, Acid, asparagine, glutamine, and aspartic acid) It is possible to modify the native sequence to replace it with a new codon that expresses Wear. New codons such as codons that express polar amino acids such as aspartic acid Numerous sequences can be created in which different codons are replaced by dons. afterwards, The resulting modified DNA sequence is expressed to obtain a modified protein. The solubility of the modified protein test. Proteins found to be soluble are tested for disease-related abnormalities Make sure that the properties are maintained.   Soluble proteins that are larger than the native protein can be made in a similar manner. Array It can be modified systematically by adding codons in the sequence at some end. Modification Express the DNA sequence to provide a longer protein than the native protein. Long protein Test for solubility. Testing for proteins that are found to be soluble and related to disease Make sure that it retains anomalous properties.   Amino acid modification, shortening, by substituting one amino acid for another, Or, in addition to elongation, repair native proteins by binding new molecular components. Decoration It is also possible. The molecular component to be added can be at either end of the native protein. Or to any part of the chain. Common parts to connect Has an "R" group that is completely different from any naturally occurring amino acid "R" group, Substitution of amino acids by alternative amino acids that improve the solubility of white matter Is included. For example, the added "R" group can be a highly hydrophilic group. Most Later, the native protein is repaired using all or any combination of the above modifications. It is also possible to decorate.                                Sequence determination   Determining the nucleotide sequence encoding a naturally occurring insoluble protein is a method of the present invention. Not part of Such sequences are known and include, for example, some mammalian Pr The sequence of the P gene is disclosed in U.S. Patent 5,565,186 issued October 15, 1996. The sequence of several β-amyloid proteins associated with Alzheimer's disease No. 5,387,746, issued Feb. 7, 2014. Nucleotide sequence information In addition to this report, this document includes information on the structural aspects of the proteins expressed by that sequence. Contains a considerable amount of information about   After obtaining the sequence of interest, make a copy using standard methods and make a copy. Later, the copies are separated into groups or pools. For example, 5 or more copies, 10 Or more, 100 or more sequence groups or pools. Each group Make this classification so that loops or pools can be manipulated separately. Preferably, Each pool contains 10 or more identical sequences.   Once a pool of identical sequences is obtained, systematically alter the sequence in each pool. You. For example, sequences in each pool can be identified by (1) removing one or more codons (codons (2) Codon addition (cod (Can be added to either end of or within the native sequence); (3) Deletion. Proteins expressed by the genetic material Depending on the information known about the quality, certain structural features of the expressed protein It is possible to systematically remove or add a portion of the DNA to be removed or added. It is possible. In this way, a shorter or longer DNA segment is provided. Segments can be deleted or added and expressed Let And proteins in different pools have different important structural features or lack Protein pools are created that differ from each other in that respect.   After expressing different nucleotide pools to obtain different protein pools, Testing different protein pools individually for solubility first and then for biological activity . Proteins with significantly higher solubility compared to native proteins should be tested further Become a candidate. Methods for determining whether a modified protein is soluble depend on the protein being tested. May vary by white matter. For example, pure water, a normal body fluid similar to human fluid Possibility to test proteins in solution or in aqueous solutions containing mild detergents is there. The solvent must be an aqueous solvent and the selected solvent will substantially Characteristic of modified proteins due to their solubility in solvents It is necessary to select something that can be attached. In some cases, The protein is soluble by centrifuging the solution at 100,000g for about 1 hour. It may be desirable to check for If the protein remains in the supernatant without precipitation, The protein can be determined to be soluble. Those skilled in the art will be able to use Appropriate other solubility tests would be readily recognizable.   Second, for the identified soluble proteins, do not digest insoluble native proteins Another test is performed, such as the ability to be digested by a particular enzyme. The protein is digested If not, additional biological tests are performed, such as dispensing and inoculating animals. Contact The sown animals are observed to determine if they develop as a result of the inoculation. Feeling Transgenic animals that develop disease after inoculation with infectious prions Transgenic animals and compounds using such transgenic animals Are hereby incorporated by reference to disclose and describe the test methods for No. 5,565,186 issued Oct. 15, 1996.                               Insoluble protein   The soluble protein of the present invention is a modification of an insoluble native protein associated with a specific disease. Things. The present invention also applies to protein modifications to be discovered in the future. But the book The invention now applies directly to known insoluble native proteins associated with certain diseases. Can be fitted. Below is a non-limiting table of insoluble proteins associated with disease .disease Insoluble protein Alzheimer's disease APP, Aβ peptide,                                           α1 antichymotrypsin,                                           tan, non-Aβ component Prion disease, Creutzfeld-Jakob disease, Scrapie and bovine spongiform encephalopathy PrP^Sc ALS SOD and neurofilament Pick disease Pick body Parkinson's disease Rui body Type II diabetes amylin Multiple myeloma-- Plasma cell disease IgL chain Familial amyloid polyneuropathy transthyretin Medullary carcinoma of the thyroid gland Procalcitonin Chronic renal failure β_TwoMicroglobulin Congestive heart failure Atrial natriuretic factor Senile heart and systemic amyloidosis transthyretin Chronic inflammation Serum amyloid A Atherosclerosis ApoA1 Familial amyloidosis gelsolin   The above insoluble proteins each include several variants that fall within the scope of the present invention. Or contain variants. PrP associated with prion disease Known pathogenic variants and polymorphisms in the gene are described below, but are Di and bovine sequences are described in U.S. Patent No. 5,565,1865, issued October 15, 1996. Have been.                                 Mutation table  In addition, the protein often has two different three-dimensional structures with the same amino acid sequence. It is important to note that there are several things. One three-dimensional structure is associated with disease features However, while generally insoluble, other structures are not associated with disease You. The present invention renders insoluble proteins soluble, but substantially all associated with the disease. The goal is to modify it to retain its features. Therefore, the present invention When an animal is inoculated with soluble protein, the animal develops disease.                             Synthesis of soluble proteins   The soluble proteins of the present invention have at least two features in common. First, these Soluble protein is much more soluble than the insoluble native protein from or based on it. High resolvability. Second, the unusual nature of the disease-related native protein continues You. The soluble proteins of the present invention can be divided into several different categories. First In addition, soluble proteins have fewer amino acids than natural insoluble proteins. is there. Other soluble proteins of the invention have more amino acids than insoluble native proteins. There are many. Yet another soluble protein has the same number of amino acids as the native insoluble protein. But with different amino acids due to some deletions or additions. In another embodiment, the native protein is combined with another agent that improves the solubility of the molecule. Chemical modification by including amino acids or attaching side chains. Finally The soluble protein of the present invention can include all the modifications described above. Insoluble heaven The soluble protein of the present invention, which has fewer, more, or different amino acids as compared to the native form Quality can be produced chemically or using molecular genetics. Test Because it is often desirable to produce a large number of different proteins first, It is generally preferred to produce such proteins using offspring genetics. Natural type Fewer or more amino acids compared to white matter, or just different amino acids Containing proteins can be produced using molecular genetics by the methods described below. it can.   A sequence that does not need to be known but produces an insoluble native protein when expressed Must be isolated. Once this genetic material is isolated, it is systematically modified. Can be For example, a DNA sequence containing 100 codons can be copied and 100 different Rules. One different codon is removed for each pool. Various alternative systematic deletion methods will be apparent to those skilled in the art. For example, Delete every 5 dongs or create codon groups at specific ends or within sequences It can be deleted.   Similarly, a DNA sequence is ligated at any desired site, and one or more Can be added. When adding codons, polar amino acids must be It is preferable to add a codon to add the amino acid to the protein. Coding for acidic polar amino acids, due to their tendency to increase the solubility of It is particularly preferred to add a don. One skilled in the art will recognize that some codon deletions Codons are added at the same time as the original codon encoding the insoluble native protein. Low number of codons or a small number compared to the sequence encoding the native protein Or modified sequences containing more or more codons. It seems to be.   As disclosed in U.S. Pat. No. 5,182,366 to Huebner et al. , Tens of thousands and tens of thousands of desirable sequences in a relatively short time One million variants can be made.   Once the desired sequence has been generated, it can be converted to the appropriate sequence using techniques well known to those of skill in the art. Put in an expression vector. Then, put the expression vector into a suitable host and allow it to express Obtain a number of different amino acid sequences. The amino acid sequence is then applied using the concepts of the present invention. To determine which sequences are soluble. Then, the soluble sequence was further added. Tested to retain and retain the disease-related abnormal properties of the insoluble native protein To determine if Soluble and has abnormal properties related to the disease The protein is the protein of the present invention.   Use chemical synthesis to create another variant, and then use the soluble and insoluble native protein Test for biological activity that retains abnormal properties associated with quality diseases You can also. Use chemical synthesis to create proteins containing uncoded amino acids Can be manufactured. In addition, it contains the D-form of the optical isomer of the naturally occurring amino acid. Thus, the amino acid sequence can be chemically synthesized. Uncoded amino The method of making the acid and its millions of variants was announced on May 30, 1995 by Rutter. ter) et al., US Patent No. 5,420,246. Using chemical method Can produce a large number of amino acid variants. One example is 19 Zuckerman et al., Published March 31, 94, in WO 94/06451. It has been disclosed. For the purposes of the present invention, when performing such a method, The "R" group of the amino acid or the replacement amino acid generally imparts a hydrophilic character to the molecule as a whole. Polar groups that provide and thereby improve the solubility properties of the molecule.   Soluble molecules can be produced using chemical synthesis methods, but such soluble Molecules may be less useful for making antibodies. Made using this molecule Antibodies may have an affinity for a site that is not present on the insoluble native protein. There is a potential. Therefore, for the purposes of the present invention, not only soluble truncated sequences, To generate antibodies that also bind to long, insoluble native sequences, the soluble "truncated" It is preferable to use an amino acid sequence.                                Antibody production   When the soluble proteins of the present invention are produced and tested for biological activity, these proteins White matter is very useful for producing antibodies that bind to soluble proteins as well as to native proteins Become. Such an antibody is first obtained from a native protein insoluble in the host animal (or (The soluble protein of the present invention). The host animal can be any animal Preferably a book, such as a mouse, rat, guinea pig, or hamster. A host animal of the type defined in the specification, most preferably a mouse. The host animal is preferably genetically distinct as defined herein. Inoculate proteins endogenous to different species. For example, transgenic mice To human PrP^ScInoculate. Thereafter, the inoculated host animal produces antibodies. Scraped It is preferable to inoculate an animal having an endogenous gene that expresses the expressed protein. example For example, a mouse in which the endogenous PrP gene has been deleted has been described in U.S. Patent No. 5,565,186. I have. A common method for deleting endogenous genes was reported by Capecchi et al. In November 1995. It is described in U.S. Patent No. 5,464,764, issued on the 7th.   After allowing the mice to produce antibodies, the mice are sacrificed and bone marrow and spleen cells are harvested. Start. Lyse cells, extract RNA, and reverse transcribe cDNA. Then, the antibody H chain And the L chain (or a part thereof) is amplified by PCR. Amplified cDNA library Or use a range of variants, It can be used after being manipulated to increase the size of the library.   Subsequently, the amplified cDNA encoding the IgG heavy chain and the amplified cDNA encoding the light chain. The obtained cDNA is converted into an H chain fragment in the first expression cassette of the vector. Is included in the second expression cassette of the vector. Phage display vector to include the cDNA insert encoding the strand fragment. The vector is inserted into an IgG phage display (eg, pComb3 vector). Create a play library.   The ligated vector is then transferred to a linear primer using methods well known in the art. Packaged in M13. Culture the E. coli packaged library Infect the nutrient solution and amplify the number of phage particles. After lysing the bacterial cells, The diparticles are isolated and used for panning (sorting).   The soluble proteins of the present invention are very valuable for panning. The protein is soluble Therefore, it is easy to contact the phage particles, You can determine if you are expressing an antibody that binds to the substance. The binding of the protein of the present invention occurs. Labels can be added to make detection easier when they come. Then the book Phage that bind to the protein of the invention are isolated and cloned. That genetic material Enter into cells and produce antibodies. Fusing cells with appropriate melanoma cells A hybridoma for producing a clonal antibody can be produced.                                   Example   The following examples demonstrate how to make soluble proteins and find such proteins. It is intended to fully disclose and explain to a person skilled in the art how to It is not intended to limit the scope of what is considered. The number to use (eg amount, temperature Efforts have been made to ensure accuracy with respect to Need to be Unless indicated otherwise, proportions are by weight and molecular weight is by weight average. Temperature is in degrees Celsius; and pressure is at or near atmospheric.                                  Example 1                             PrP segment removal   In certain embodiments, segments of the PrP molecule have been systematically removed. Specifically Is PrP^CRegion in which a secondary structure is assumed (Z. Huang et al., Proc. Natl. Acad. Sci. . USA 91, 7139-7143 (1994)). 4 putative helics If you delete each of the^ScFormation was prevented. But helix 2 and 3 Removing the intervening 36 residue loop had no effect. Resulting PrP^Sc The molecule has an additional sequence of an N-terminal signal peptide and a C-terminal glycolipid anchor. Contains 106 amino acids after cleavage, so PrP^ScNamed 106. PrP^Sc106 is Resistant to digestion by proteinase K, but soluble in 0.5% sarkosyl Was. PrP^ScWith the discovery of 106, PrP^ScStudy of Structure and PrP^ScResearch on formation mechanism Ultimate and PrP^ScSpecific anti Body production becomes very easy.   PrP^CFrom PrP^ScIn the process of conversion to PrP, it undergoes a major structural change (KM Pan, et al.). Proc. Natl. Acad. Sci. USA 90, 10962-10966 (1993)). In this structural change, Insolubility in non-denaturing detergent and acquisition of resistance to proteinase K digestion (S.B. Prusiner et al., Biochemistry 21, 6942-6950 (1982); R.K. Meyer et al., Proc. Natl. Acad. Sci. USA 83, 2310-2314 (1986)). PrP^ScThe study of the structure of This molecule has been restricted due to its insolubility (SB Prusiner et al., Cell 35, 3). 49-358 (1983); B.W. Caughey et al., Biochemistry 30, 7672-7680 (1991); Gasset Proc. Natl. Acad. Sci. USA 90, 1-5 (1993); Safar et al. Biol. Chem. 268, 20276-20284 (1993)). But PrP^CSynthesis of PrP analogs similar to Recombinant fragments were easier to study structurally (H. Zhang et al., J. Mol. Biol. 250, 5). 14-526 (1995); Mehlhorn et al., Biochemistry 35, 5528-5537 (1996); Riek et al. Nature 382, 180-182 (1996)). Research is not only insoluble, but also It is difficult to express high levels of recombinant PrP (I. Mehlhorn et al., Biochemistry 35, 5528-5537 (1996); M. Scott et al., Protei n Engineering 2, 69-76 (1988)).                                 Example 2                            PrP cloning   To study the structural features of mammalian PrP by molecular modeling, chicken P Molecular cloning of the cDNA encoding rP was performed. Several types of structural prediction algorithms Using the system, four regions presumed to be secondary structures were identified (Z. Huang et al., Proc. Natl. Acad. Sci. USA 91, 7139-7143 (1994); Gasset et al., Proc. Natl. Acad. Sci. USA 89, 10940-10944 (1992)). Specific area is α helix or β sheet Although there was a disagreement about whether or not to take the structure, all analyzes It was predicted that the region would have a secondary structure. Therefore, H1, H2, H3 and H4 and life Synthetic peptides corresponding to each of the four named regions were made. In aqueous buffer In H1, H3 and H4 unexpectedly adopt a β-sheet structure (M. Gasset et al., Proc. N atl. Acad. Sci. USA 89, 10940-10944 (1992)).                                   Example 3                                 Structural analysis   When H1 of β sheet structure and H2 of coil were mixed, H2 was converted to β sheet (J . Nguyen et al., Biochemistry 34, 4186-4192 (1995)). More than 56 residues including H1 and H2 Long peptides adopt an α-helix structure in an aqueous buffer, and α-helix in the H1 region. (H. Zhang et al., J. Mol. Biol. 250, 514-526). (1995)). The secondary structure in the H2 region as determined by NMR has become less clear. 11 NMR studies of the single residue C-terminal fragment identified the H3 and H4 regions (R. Riek Et al., Nature 382, 180-182 (1996)). Also, in these studies, between H2 and H3 The short α-helix in the loop, and residues 128-131 and 161-164. A short anti-parallel β-sheet was identified.   PrP^CBased on the molecular model and CD and FTIR tests of PrP^C The molecule is PrP^ScA systematic test of the model was conducted for its ability to convert to. The exam is Performed on both cultured neuroblastoma (N2a) cells and transgenic (Tg) mice. As a result, PrP^ScPutative secondary structure for the need for each region to support the formation of An area map was obtained (Figure 1).                                 Example 4                           Epitope tag-antibody   Previously MHM2 PrP in ScN2 cells^ScEpitope tag that has been shown to convert to MHM2 PrP with^CThe molecule was used to demonstrate in scrapie infected N2a cells (ScN2a). (A. Taraboulos et al., Proc. Natl. Acad. Sci. USA 87, 8262-8266 (19). 90); Rogers et al., Immunol. 147, 3568-3574 (1991); Scott et al., Protein Sci . 1, 986-997 (1992)). All PrP constructs expressed in ScN2a cells were transferred to pSPOX.ll The cells were cloned into the neo vector and cells were transiently transfected (M.R. . Scott et al., Protein Sci. 1, 986-997 (1992)).   Epitope-tagged MHM2 PrPs are the 108 and 111 found in SHa and Hu PrP. It has two Met residues at the number position. This epitope is recognized by α-PrP 3F4 mAb (M. Rogers et al., Immunol. 147, 3568-3574 (1991); R. J. Kascsak et al., J. V. irol. 61, 3688-3693 (1987)). PrP encoded by MHM2^CIs in ScN2a cells Easy with PrP^ScTo evaluate the effect of selective deletion of specific regions in the PrP molecule. Value (Figure 1).                                 Example 5                           Expression in ScN2a cells   Using MHM2 in which the N-terminal region consisting of residues 23 to 88 has been deleted as a starting material Was. This construct contains an α-PrP 3F4 mAb reactive protein that is resistant to proteinase digestion. PrP in ScN2a cells as shown by production^ScSupported the formation (M. Rogers et al., Proc. Natl. Acad. Sci. USA 90, 3182-3186 (1993)). MoPrP After removing residues 33-80, the resulting construct is PrnP^0/0Mo Scray Mouse It has been shown to be sensitive to peplion (M. Fischer et al., EMBO J. 15).   Three regions in the putative secondary structure, designated H2, H3 or H4, have been deleted. Another structure N-terminally truncated MHM2 PrP construct wherein residues 23-88 have been deleted in the construction The H2, H3 and H4 regions were deleted. Each of the deleted PrPs was used in ScN2a cells. Was expressed. H2 (residues 122-140), H3 (residues 177-200) and H4 (residues 201-217) ) The α-PrP 3F4 mAb immunostaining of each construct with the region deleted and expressed in ScN2a cells Detected by Does not include both consensus sites for Asn-linked glycosylation H3 deletion (C. Locht et al., Proc. Natl. Acad. Sci. USA 83, 6372-6276 (198 6)) M_rSeparated as a single band of 1919 kDa. Two other structures also launched Revealed: one deleted loop containing 36 residues (141-176) between H2 and H3 Another is the construction of a disulfide bond between residue 178 and Cys213. Has a point mutation in which residue 178 has been replaced with Cys to Ala to prevent formation It is a construct to do.                                 Example 6                             Proteinase K resistance   Judgment based on resistance to digestion by proteinase K (20 µg / ml, 30 minutes, 37 ° C) However, due to deletion of the H2, H3 and H4 regions and mutation of Cyc178, PrP^Scof Formation was prevented. From this result, MHM2 was an N-terminal deletion in which residues 23 to 88 were deleted. Like the offspring, it was shown that the protein was converted to a protease-resistant isoform.   Deletion of the loop between H2 and H3 containing 36 residues (141-176), It did not prevent the conversion to an anti-serum isoform. PrP^ScProteases during formation Resistance Acquisition appears to occur within the caveolae-like region (CLD), and PrP^CIs the GPI anchor More directed to CLD (A. Taraboulos et al., J. Cell Biol. 129, 121-132 (1995)) . Cells expressing the construct lacking the loop were contacted with PIPLC. as a result MHM2 (23-88, 141-176) (Deleted) have been shown to be released by this enzyme (N. Stahl et al.). Cell 51, 229-240 (1987)). This is because PrP106 moves from the ER to the cell surface, PrP^CIt also indicates that it is bound there by a GPI anchor.                                 Example 7                               (STE) area removal   Analysis of deletion of H1 including residues 108-121 detected by α-PrP 13A5 mAb To create a possible SHaPrP epitope, a 1138M mutation was created and performed (M. Ro gers et al. Immunol. 147, 3568-3574 (1991); R.A. Barry et al. Infect. Dis. 1 54, 518-521 (1986)). Similar to the deletion of H2-H4, removal of H1^ScFormation is impeded I was stopped. Then, using α-PrP 13A5 mAb, using canine pancreatic microsomal membrane Adjacent to H1 has been implicated in regulating the transport of PrP synthesized in a cell-free system The deletion of the transcription termination effector (STE) region consisting of residues 95-107 of S. Yost et al., Nature 343, 669-672 (1990)). As with the H1-H4 deletion, the deletion of the STE region Also PrP^ScFormation was prevented.   Based on the above results, PrP^ScLimited digestion of 106 with proteinase K (limited digestion), then M_rThe change in value was examined. In the absence of proteinase K , MHM2 PrP encoded by the transfected construct^CAnd PrP^Sc Both isoforms were immunostained with the αPrP 3F4 mAb. M_rWide value Probably due to aberrant Asn bond glycosylation . After limited proteolysis, M_rThe value decreases and the molecular M_rThe range of values has been reduced. M_rValue of According to the changes, PrP during limited proteolysis^ScSome amino acids from the 106 N-terminus There is a high possibility that the acid residues have been removed. Micro-sequencing solves this problem Can be solved.                                 Example 8                                  Solubility   The insolubility in non-denaturing detergents is due to protease-resistant PrP^ScWith the immutable characteristics of (R.K. Meyer et al., Proc. Natl. Acad. Sci. USA 83, 2310-2314 (1986); S.B. Prusiner et al., Cell 35, 349-358 (1983)). Because this is known, PrP106 is expressed Increase the concentration of N-lauroyl sarcosine (Sarcosyl) in ScN2a cells Dissolved in the solution. The suspension was centrifuged at 100,000 × g for 1 hour at 20 ° C. 0. At concentrations above 5% sarkosyl, all PrP^Sc106 was found in the supernatant. α-PrP MoPrP by heron polyclonal RO73 antiserum^ScBlot again for detection When probed, all immunostaining was found in the precipitated fraction and in the supernatant. I couldn't.                                 Example 9                                 Structural analysis   The results obtained show four putative helical regions identified by molecular modeling. And the STE region found by cell-free translation experiments^ScNecessary for formation It indicates that there is. Also, from this result, PrP^CAnd PrP^ScFound in both Disulfide bond between Cys residues 178 and 213 (E. Turk et al., J. Bioch. em. 176, 21-30 (1988))^ScIs essential for the formation of This When the sulfide bond is broken by the C178A mutation, this mutant PrP^CPrP^ScTurn to Although the exchange rate was reduced, the fraction highly enriched for scrapie infectivity was treated with 2% β-mercapto. Syrian hamsters can be treated with ethanol or 100 mM dithiothreitol. Did not decrease the prion titer measured by the bioassay in S.B. siner et al., Biochemistry 19, 4883-4891 (1980)).   Prp27-30 (PrP^CRecombinant P corresponding to the residue found in the protease resistant core When rP (90-231) was purified under denaturing conditions and regenerated, disulfide bond After formation, mammalian PrP^CA structure containing a highly α-helix similar to that of (I. Meh lhorn et al., Biochemistry 35, 5528-5537 (1996)). This disulfide bond forms a C It is believed to stabilize the terminal α-helix, H3 and H4 (Z. Huang Proc. Natl. Acad. Sci. USA 91, 7139-7143 (1994); Huang et al., Folding &  Design 1, 13-19 (1996)). This is a recombinant PrP (121-231) expressed in E. coli. NMR structural analysis of the C-terminal fragment of (R. Riek et al., Natur e 382, 180-182 (1996)). N-terminal 99 amino acids and Asn-linked CHO and GPI RPrP without anchor (121-231) is PrP^CAccurately reflects the structure of the C-terminal region of It is unknown whether it will. However, from the analysis data of rPrP (121-231), H3 and It was shown that an α helix was predicted in the H4 region.                                 Example 10                               Features of PrP106   PrP106 without 23-88 residues and 141-176 residues, PrP^ScLike a molecule You. This is due to two antiparallel β-strands consisting of residues 128-131 and 161-164. The short region of the β sheet formed by^ScIs unlikely to be a starting point for Supports the view (R. Riek et al., Nature 382, 180-182 (1996)). Furthermore, PrP^CRP The third alpha helix (144-154) found in the estimation loop of rP (121-231) is not It was predicted to have a stable structure, but this was also used to gain resistance to limited proteolysis. Is not necessary.   The results shown here are for PrP^ScProtease resistance and insolubility separated for the first time Things. From such results, purified PrP^ScAggregation that should contribute to the insolubility of The view that this is not necessary for protease resistance is supported.   Partially denatured PrP^CPrP^ScRadiation from a recent cell-free mixing experiment Sex-labeled PrP^CThe protease resistance indicated by PrP^ScInseparable from binding to aggregation (D.A. Kocisko et al., Nature 370, 471-474 (1994); B. Caughey Et al., Jr. Chem. Biol. 2, 807-817 (1995)). However, the results herein show that insoluble And protease resistance are shown to be separable. These results are PrP27-30 in dendritic aggregates dispersed in liposomes and resistant to proteolytic digestion (R. Gabizon et al., J. Biol. Chem. 263, 4950-4). 955 (1988)).   PrP shown^Sc106 is full length PrP^CLower solubility than full-length PrP^ScYo Was shown to be much more soluble. This is PrP^Sc106 molecules (or PrP^{S c} Interaction between the 106 protease-resistant units)^Sc Suggests that they are more easily destroyed by surfactants than by intermolecular interactions I have.   Using Prg-depleted Tg mice with background, PrP^ScFormation of 106 To By examining the PrP^ScIf 106 can be confirmed to support prion infectivity Seem. Furthermore, PrP^Sc106 is amyloidogen in vivo and in vitro Experiments to determine whether or not to support infectivity show that the difference between infectious and amyloidogenic Separation may be shown to be associated with the solubility of this molecule.   PrP^ScPurification of the 106 intermediate is straightforward due to its unique solubility. First PrP with weak surfactant^Sc106 is separated from PrP106, then strong surface activity With other PrP^ScSeparate from molecules. Protease from purification protocol Omitting digestion can lead to structures that can be destroyed by the presence of proteases. This is advantageous for structural analysis.                                 Example 11                                Antibody formation   ScN2a is a protease-resistant but soluble PrP^ScBecause it can form molecules, fur PrP with display^ScIsolation of specific antibodies is straightforward. Specifically, PrP^Sc Antibody discovery, soluble PrP^ScPanning for soluble compounds like 106 molecules It is very easy to do the screening (R.A. Williamson et al., Pro. c. Natl. Acad. Sci. USA 93, 7279-7282 (1996)). PrP^ScBecause 106 is available And PrP^ScThe determination of the tertiary structure of the substance should progress substantially, along with PrP^CIs Pr P^ScThe elucidation of the mechanism of the transition to Iraq should be greatly advanced. ScN2a cells PrP^ScThe formation of a soluble form of Development is facilitated (FE Cohen et al., Science 264, 530-531 (1994)). Furthermore, Pr Soluble proteins of the present invention, such as P106 and variants thereof, are PrP^ScOf diseases related to One can test its own effect of slowing progression. Prion disease is biology And unprecedented in medicine, the design of drugs to treat these diseases Is PrP^ScIt is necessary to understand in detail the structural changes that PrP undergoes as well as the formation of It is likely. Bovine prions infect humans in the UK and France and induce vCJD (G. Chazot et al., Lancet 347, 1181 (1996); R.G. Will et al.) , Lancet 347, 921-925 (1996)), and the development of effective treatments for prion diseases is very important. It became important.   When producing an antibody using the soluble protein of the present invention, insoluble natural protein It is generally preferred to use a soluble protein with fewer amino acids than quality. small By using a small protein, it has a binding affinity for the insoluble native protein In the sense, the production of antibodies that bind to added amino acids, which are inappropriate antibodies Can be avoided.   The invention shown and described herein is believed to be the most practical and preferred embodiment. It is what is done. However, departures from that embodiment are also possible, and It will be appreciated that modifications will be apparent to those of skill in the art upon reading It seems to be that.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 25/28 C07K 16/18 C07K 16/18 G01N 33/15 Z C12N 15/09 33/50 Z G01N 33 / 15 33/53 D 33/50 C12N 15/00 A 33/53 A61K 37/02 (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE) , LS, MW, SD, SZ, UG), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, B , BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR , TT, UA, UG, UZ, VN, YU (72) Cohen Fred E. Inventor San Francisco Rhode Island Street 767, California, USA (72) Muramoto Tamaki, United States San Francisco Buckingham Way # 902 265

Claims (1)

[Claims] 1. (A) deletion of amino acids from the natural amino acid sequence; (b) addition of amino acids to the natural amino acid sequence; (c) binding of hydrophilic molecular components to the natural amino acid sequence; and (d) (a) to (c) produced by a method selected from the group consisting of any combination of the above, a soluble form of a protein that is insoluble in a natural form, wherein the produced soluble protein is an insoluble natural form protein associated with a disease. A soluble form of a protein that retains its properties. 2． 2. The soluble protein according to claim 1, which is produced by a method comprising the steps of: obtaining a DNA sequence encoding a native protein having an insoluble structure; making a copy of the DNA sequence; Removing a plurality of different codons from a plurality of DNA sequences encoding a protein to provide a plurality of different DNA sequences in which the removed codons are different from each other; expressing the different DNA sequences to obtain a plurality of different proteins Testing the solubility and biological activity of the protein; and isolating a protein having substantially the same biological activity as the native protein in terms of higher solubility and disease-related properties than the native protein. A step in which a native protein having an insoluble structure is selected from the group consisting of PrP ^Sc protein; β-amyloid protein and Lewy bodies. 3． Natural insoluble forms of the protein are found in mammals with Alzheimer's disease, prion disease, Creutzfeldt-Jakob disease, scrapie and bovine spongiform encephalopathy, ALS, Pick's disease, Parkinson's disease, type II diabetes, multiple myeloma--plasma Cell disease, familial amyloid polyneuropathy, medullary carcinoma of the thyroid gland, chronic renal failure, congestive heart failure, senile heart and systemic amyloidosis, chronic inflammation, atherosclerosis, familial amyloidosis 2. The protein according to claim 1, which induces a disease of the central nervous system which is a disease of the present invention. Four. Determining a DNA sequence encoding a native protein having an insoluble structure; making a copy of the DNA sequence; adding a plurality of different codons to a plurality of DNA sequences encoding the native insoluble protein; Providing a plurality of different DNA sequences wherein the different codons differ from each other; expressing the different DNA sequences to obtain a plurality of different proteins; testing the solubility and biological activity of the different proteins; and a native protein The soluble protein of claim 1, wherein the protein is produced by a method comprising isolating a protein having substantially the same biological activity as the native protein with respect to higher solubility and disease related properties. natural proteins with structures, APP, a [beta] peptide, [alpha] 1-antichymotrypsin, tan, non-a [beta] component, PrP ^Sc, SOD and neurofilament pick Body, Ryui bodies, amylin, IgL chain, transthyretin, procalcitonin, beta ₂ microglobulin, atrial natriuretic factor, serum amyloid A, ApoA1, soluble protein selected from the group consisting of gelsolin. Five. A modified PrP ^Sc protein that is digested by a protease and has a solubility that is at least twice that of a native PrP ^Sc . 6． It is five times more soluble than native PrP ^Sc and is further characterized by causing disease in mammals, and further characterized by having no loop structure that is present in native PrP ^Sc and generally contains about 36 amino acid residues. 6. The modified PrP ^Sc protein according to claim 5, wherein 7． Protein is PrP ^Sc 106, claim 5 modified PrP ^Sc protein as claimed. 8． An antibody that selectively binds to the soluble protein according to claim 1. 9． Contacting the sample with the antibody of claim 8; and measuring the amount of binding between the antibody and the sample to determine whether insoluble native protein associated with the disease is present in the sample. A method of testing a sample for the presence of an insoluble native protein associated with a disease, comprising the steps of: Ten. Dissolving the soluble protein of claim 1 to form a solution; contacting the solution with the drug; and determining the effect of the drug on the properties of the soluble protein associated with disease. Screening method.